Custom qBiomarker Copy Number PCR Arrays are designed to measure the number of copies for a genomic loci panel of your choice. Assays are available for every 200 base pairs covering the entire human genome. Custom qBiomarker Copy Number PCR Arrays are highly suitable for verification of single nucleotide polymorphism (SNP) array or array-comparative genomic hybridization (aCGH) data, and are available in plate or Rotor-Disc formats.

To order a Custom qBiomarker Copy Number PCR Array, please use this excel file.

The 96-well plate format provides a custom panel of copy number assays, and includes the MRef assay in quadruplicate.

qBiomarker Copy Number PCR Array, 384-well plate format.

The 384-well plate format provides a custom panel of copy number assays, including the MRef assay.

qBiomarker Copy Number PCR Array, 100-well Rotor-Disc format.

For use with Rotor-Gene real-time cyclers, the Rotor-Disc format provides a custom panel of copy number assays and includes the MRef assay in quadruplicate.

qBiomarker Copy Number PCR Assays accurately identify aneuploidy.

A qBiomarker Copy Number Assays designed to target AR, which is on the X-chromosome, was tested against 4 cell line DNAs containing 1 copy (XY, Coriell NA13619), 2 copies (XX, Coriell NA01921), 3 copies (XXX, Coriell NA03623) and 4 copies (XXXX, Coriell NA11226) of X-chromosome. Chromosomal aberrations had been previously identified by cytogenetic methods. A control assay, targeting a stable, multi-copy region in the human genome, was used to normalize the amount of DNA input. The ΔΔCT method was used to calculate the gene copy number, using XX (Coriell NA01921) as a 2-copy reference. The assay was tested against each sample in quadruple replicate reactions, and a t-test was performed.

qBiomarker Copy Number PCR Assays accurately identify aneuploidy.

A qBiomarker Copy Number Assay designed to target MECP2, which is on the X-chromosome, was tested against 4 cell line DNAs containing 1 copy (XY, Coriell NA13619), 2 copies (XX, Coriell NA01921), 3 copies (XXX, Coriell NA03623) and 4 copies (XXXX, Coriell NA11226) of X-chromosome. Chromosomal aberrations had been previously identified by cytogenetic methods. A control assay, targeting a stable, multi-copy region in the human genome, was used to normalize the amount of DNA input. The ΔΔCT method was used to calculate the gene copy number, using XX (Coriell NA01921) as a 2-copy reference. The assay was tested against each sample in quadruple replicate reactions, and a t-test was performed.

RNase P and other single-copy genes are not suitable normalizers for sample input.

Genomic DNA samples with many amplifications and/or deletions, such as tumor DNA, require users to verify their single-copy reference genes to ensure that the reference genes themselves have not undergone a change in copy number. The advantage of a multi-copy reference assay is demonstrated in the measurement of the relative copy number of RNase P in 2 common cancer cell lines. The absolute average copy numbers of RNase P per normal genome copy amount of DNA were determined in 2 breast cancer cell line (SKBR3 and MCF7) genomic DNAs with the ΔΔCT method, using qBiomarker Multi-Copy Reference Copy Number PCR Assay as the normalization control of DNA input. The absolute copy number of RNase P per normal genome in reference genomic DNA is assumed to be 2. The copy number of RNase P in both SKBR3 and MCF7 is significantly altered, demonstrating that this reference gene would be an unreliable normalizer for these samples.

The MRef assay provides superior normalization for copy number determination.

Tumor cell line DNA (SKBR3) and reference genomic DNA (Promega) were mixed in different ratios (100%, 87.5%, 75%, 50%, 25%, 12.5%, and 0% SKBR3 cells), and the DNA mixes were tested for the gene copy number of GRB7 (a gene that is significantly amplified in SKBR3), using either the MRef assay (top) or the RNase P assay (bottom) as the reference. The GRB7 copy number in reference DNA is assumed to be 2. The expected GRB7 gene copy numbers in 87.5%, 75%, 50%, 25%, and 12.5% mixing ratio samples are calculated based on the GRB7 gene copy number in 100% SKBR3 gDNA sample, and the mixing ratio between the SKBR3 gDNA and reference genomic DNA.

Performance

When using real-time PCR to evaluate copy number changes in DNA samples, two elements are critical: real-time PCR assay performance, and reliable normalization of DNA input. Every qBiomarker Copy Number PCR Assay on a Custom qBiomarker Copy Number PCR Array is wet bench-tested for several characteristics affecting the accuracy of real-time PCR results: specificity, wide dynamic range, and uniformly high amplification efficiency. Laboratory verification of assay quality ensures that qBiomarker Copy Number PCR Assays deliver reliable results.

Indeed, qBiomarker Copy Number PCR Assays accurately identified aneuploidy in cell lines containing chromosomal aberrations previously identified by cytogenetic methods. Assays for AR and MECP2, which are both on the X chromosome, correctly quantified gene copy number in cell lines with 1, 3, and 4 copies of the X chromosome.

Custom qBiomarker Copy Number PCR Arrays use the qBiomarker Multicopy Reference Copy Number PCR Assay (MRef) to provide superior normalization of DNA input. Single-copy reference genes such as RNase P can yield unreliable normalization, as in cancer cell lines. By contrast, the MRef assay provides accurate normalization. Together, the laboratory verification of each qBiomarker Copy Number PCR Assay on the array and the superior DNA input normalization provided by the MRef assay ensure accurate, reliable results.

Principle

Each Custom qBiomarker Copy Number PCR Array contains a panel of qBiomarker Copy Number PCR Assays for GOIs or ROIs of your choice. Four replicates of each assay are included to increase the accuracy of copy number calls using statistical analysis. The arrays are available in 96-well plate, 384-well plate, and Rotor-Disc formats, all of which are designed to your specifications, to fit the needs of your experiment.

All qBiomarker Copy Number PCR Assays are designed in unique regions of the genome. A multicopy reference assay, the qBiomarker Multicopy Reference Copy Number PCR Assay (MRef) is included on each array, providing superior normalization for DNA input compared to single-copy reference genes. The reference assay recognizes a stable sequence that appears in the human genome over 40 times, and whose copy number is not affected or minimally affected by local genomic changes. Inclusion of this reference assay during testing allows use of the ΔΔCT method to accurately make copy number calls or relative copy number change calls for specific targets.

The simplicity of the qBiomarker Copy Number PCR Array format and operating procedure allows routine copy number profiling in any research laboratory with access to real-time PCR instruments.

Procedure

To complete the qBiomarker Copy Number PCR Array procedure, start with genomic DNA isolated from fresh or frozen samples, or DNA from FFPE sections (QIAGEN QIAamp DNA Mini Kit or FFPE Tissue Kit is recommended). Optionally, if DNA sample quantity is limited, DNA from fresh or frozen tissues can be uniformly amplified using QIAGEN REPLI-g UltraFast Kit. Then, mix your DNA with the appropriate qBiomarker SYBR® Green Mastermix and aliquot the mixture into each well of the same qBiomarker Copy Number PCR Array plate containing predispensed locus-specific primer assays. Real-time PCR is used to determine the copy number status of a particular sample using the ΔΔCT method by comparing the test sample with a reference genome. An optional DNA sample quality control step can be performed immediately before the detection array setup.

Applications

Custom qBiomarker Copy Number PCR Arrays are highly suited for accurate profiling of copy number alterations or variations in a custom panel of genes, such as for verification of single nucleotide polymorphism (SNP) array or array-comparative genomic hybridization (aCGH) data.